Estrogen and progesterone receptors are known to be valuable in predicting endocrine responsiveness and aggressiveness of breast cancers. Cytochemical or flow cytometric detection of these receptors in the heterogeneous cell populations found in most tumors would offer new information on heterogeneity and its significance for these predictions, as well as making the determination faster and requiring less tissue, and providing tools for basic receptor studies. We have previously shown that, although anti-receptor antibody methods appear to be valid, most existing cytochemical methods claimed to detect receptor probably do not actually do so. Because cytochemical methods offer direct detection of the steroid binding sites even in living cells, we have undertaken to synthesize new, more stable conjugates of estrogens and progestins with fluorophores, to replace the unstable, low affinity conjugates presently available. We have already developed stable 17alpha estrogen linkages of reasonable affinity, and here propose to produce fluorescein and dimethoxycoumarin conjugates based on this linkage, while also developing parallel 7alpha and 16alpha linkages for selection of the optimum ER conjugate. We have also developed stable 17alpha progestin linkages, and now propose to synthesize fluorescent conjugates for PgR based on these. The affinity increase on adding a 7alpha methyl to these progestin conjugates will be determined, and compared with the affinity of 17alpha-ethinyl-7alpha conjugates. The optimum fluorescent conjugates for ER and PgR will be rigorously selected by stability and receptor affinity. As potentially useful conjugates are generated, they will be used to develop a method for receptor detection by cytochemistry. Validation of the method will require comparison of the cytochemical results in a series of human breast tumor specimens with standard biochemical and immunohistochemical results. The new conjugates will also be used to develop a method for flow cytometric receptor detection in suspended cells or fixed suspended nuclei; this method will also be validatee by comparison with standard assays. Finally, the ability of the conjugates to cause specific responses in living cell cultures will be used to show uptake and activity, which would mean that the conjugates were suitable candidates for fluorescence polarization studies of the state of receptor-bound steroids in the target cell.